Inhibition of ammonia monooxygenase from ammonia oxidising archaea by linear and aromatic alkynes.

Ammonia monooxygenase (AMO) is a key nitrogen transforming enzyme belonging to the same copper-dependent membrane monooxygenase family (CuMMO) as the particulate methane monooxygenase (pMMO). The AMO from ammonia oxidising archaea (AOA) is very divergent from both the AMO of ammonia oxidising bacteria (AOB) and the pMMO from methanotrophs and little is known about the structure or substrate range of the archaeal AMO. This study compares inhibition by C2-C8 linear 1-alkynes of AMO from two phylogenetically distinct strains of AOA, “Candidatus Nitrosocosmicus franklandus” C13 and “Candidatus Nitrosotalea sinensis” Nd2, with AMO from Nitrosomonas europaea and pMMO from Methylococcus capsulatus (Bath). An increased sensitivity of the archaeal AMO to short-chain-length alkynes (≤C5) appeared to be conserved across AOA lineages. Similarities in C2-C8 alkyne inhibition profiles between AMO from AOA and pMMO from M. capsulatus suggested that the archaeal AMO has a narrower substrate range compared to that of N. europaea AMO. Inhibition of AMO from “Ca. Nitrosocosmicus franklandus” and N. europaea by the aromatic alkyne phenylacetylene was also investigated. Kinetic data revealed that the mechanism by which phenylacetylene inhibits “Ca. Nitrosocosmicus franklandus” and N. europaea is different, indicating differences in the AMO active site between AOA and AOB. Phenylacetylene was found to be a specific and irreversible inhibitor of AMO from “Ca. Nitrosocosmicus franklandus” which does not compete with NH3 for binding at the active site. Importance Archaeal and bacterial ammonia oxidisers (AOA and AOB) initiate nitrification by oxidising ammonia to hydroxylamine, a reaction catalysed by ammonia monooxygenase (AMO). AMO enzyme is difficult to purify in active form and its structure and biochemistry remain largely unexplored. The bacterial AMO and the closely related particulate methane monooxygenase (pMMO) have a broad range of hydrocarbon co-oxidation substrates. This study provides insights into the AMO of previously unstudied archaeal genera, by comparing the response of the archaeal AMO, a bacterial AMO and pMMO to inhibition by linear 1-alkynes and the aromatic alkyne, phenylacetylene. Reduced sensitivity to inhibition by larger alkynes suggests that the archaeal AMO has a narrower hydrocarbon substrate range compared to the bacterial AMO, as previously reported for other genera of AOA. Phenylacetylene inhibited the archaeal and bacterial AMO at different thresholds and by different mechanisms of inhibition, highlighting structural differences between the two forms of monooxygenase.